Ethanol and other alcohols may be used to fuel automobiles and other machinery powered by internal combustion engines, either as a neat fuel or as a blend with gasoline at various concentrations. For example, the use of oxygenated materials in gasoline can reduce the emission of carbon monoxide, a harmful pollutant, into the air. Among several oxygenates currently used for boosting the oxygen content of gasoline, ethanol has the highest oxygen content. The United States Environmental Protection Agency (EPA) has shown that gasoline blended with 10% ethanol reduces carbon monoxide emissions by about 25% to 30%.
Up to now, the feedstock used for the production of industrial alcohol by fermentation contain six carbon sugars and starches such as that from sugar cane, beets, corn or other edible crops. However, these agricultural crops have generally been too expensive to be used as feedstock for the large-scale production of fuel ethanol. In addition, the edible crops can only be produced in rich farm land and are in limited supply. Since the population of the world continues to increase, crops are needed to feed the people.
Plant biomass is an attractive feedstock for ethanol-fuel production by fermentation because it is renewable, and available at low cost and in large amounts around the world. The major fermentable sugars from cellulosic materials are six-carbon sugars such as glucose and five-carbon sugars such as xylose. Glucose and xylose are the major sugars present in all types of cellulosic biomass (trees, grasses, straws, etc.) with the ratio of glucose to xylose being approximately 2 to 1. The most desirable fermentations of cellulosic materials would, of course, completely convert both glucose and xylose to ethanol. Unfortunately, even now there is not a single natural known microorganism capable of fermenting both glucose and xylose effectively and efficiently to ethanol.
Some yeasts, particularly of the genus Saccharomyces, have traditionally been used for fermenting glucose-based feedstock to ethanol, and they are still the best microorganisms for converting glucose to ethanol. However, these glucose-fermenting yeasts have been found not only unable to ferment xylose but also unable to use the pentose sugar for growth. Nevertheless, glucose-fermenting yeasts can use xylulose for growth and fermentation, albeit with varying efficacy. For example, S. cerevisiae ferments xylulose very poorly while species of Schizosaccharomyces does so quite effectively. However, the latter yeast has not been used traditionally for ethanol production, particularly for large scale industrial ethanol (fuel ethanol) production.
Even though the glucose-fermenting yeasts are unable to use xylose both for growth and fermentation, there are many other natural yeasts that can use xylose for growth aerobically, but they cannot ferment xylose efficiently to ethanol. Particularly, these xylose-fermenting yeasts also ferment glucose very poorly to ethanol. These xylose-utilizing yeasts rely upon two enzymes—xylose reductase and xylitol dehydrogenase—to convert xylose to xylulose. These yeasts are different from most bacteria which rely on a single enzyme—xylose isomerase—to convert xylose directly to xylulose. The yeast xylose reductase and xylitol dehydrogenase also require cofactors for their actions; xylose reductase depends on NADPH as its cofactor and xylitol dehydrogenase depends on NAD as its cofactor. On the contrary, bacterial xylose isomerase requires no cofactor for direct conversion of xylose to xylulose.
Historically, since the early 1970s, efforts were devoted in an attempt to find new yeasts capable of effectively fermenting both glucose and xylose to ethanol in a cost effective manner. However, no ideal yeast able to ferment both glucose and xylose effectively was found by 1980.
Among xylose-fermenting yeasts, three species, Pachysolen tannophilus, Candida shehatae, and Pichia stipitis have been extensively characterized. P. stipitis and C. shihatae ferment xylose better than the other xylose-fermenting yeasts. Nevertheless, even the best xylose-fermenting yeasts lack high efficiency in fermenting xylose, and are also highly ineffective in fermenting glucose.
By 1980, scientists worldwide believed that an ideal C5/C6 co-fermenting yeast could be created by using the then-newly developed recombinant DNA techniques to engineer Saccharomyces yeast so that the resulting yeast may efficiently ferment sugars extracted from cellulosic biomass. Initial efforts were concentrated on cloning a xylose isomerase gene into yeast to render it capable of converting xylose directly to xylulose without dependence on cofactors. However, these efforts have been unsuccessful initially because the genes encoding various bacterial xylose isomerases are incapable of directing the synthesis of an active enzyme in S. cerevisiae. 
Subsequently, efforts toward genetically engineering yeasts, particularly S. cerevisiae, to ferment xylose have been focused on cloning genes encoding xylose reductase and xylitol dehydrogenase. S. cerevisiae and other glucose-fermenting yeasts do not contain any detectable xylose reductase or xylitol dehydrogenase activities, but all seem to contain xylulokinase activity. Thus, the glucose-fermenting yeasts can all ferment xylulose, but do so with varying efficacy.
Initially, researchers have only tried to clone both the xylose reductase and the xylitol dehydrogenase gene in S. cerevisiae. However, these genetically engineered yeasts still cannot effectively ferment xylose. For example, these yeasts have been incapable of fermenting more than 2% xylose. In addition, they produce large amounts of xylitol from xylose, which diverts the valuable xylose substrate from the desired fermentation path to ethanol. Nevertheless, this has been changed due to the technologies provided by the following patents: U.S. Pat. Nos. 5,789,210, 7,527,927 and 8,652,772, each to Ho et al. The methods described in these patents have made it possible to develop the glucose/xylose co-fermenting yeast that can effectively co-ferment glucose and xylose to ethanol. The yeasts developed by Ho et al. according to these patented technologies have proven to be particularly efficient for co-fermenting both glucose and xylose to ethanol. The first such glucose and xylose co-fermenting yeast was developed before 1993. The strain was designated as 1400 (LNH-ST). Subsequently, quite a few of such strains were developed by Ho et al., including strain 424A(LNH-ST), which may be abbreviated herein and accompanying drawings as 424A. As shown in FIG. 1, the 424A(LNH-ST) yeast has been proven able to produce high concentrations of ethanol when high concentrations of glucose is available. This is because the 424A(LNH-ST) yeast was developed by selecting the best ethanol producing yeast to develop the glucose/xylose co-fermenting yeast. Sedlak et al. (2004), “Production of Ethanol from Cellulosic Biomass Hydrolysates Using Genetically Engineered Saccharomyces Yeast Capable of Cofermenting Glucose and Xylose,” APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 113-116:403-16.
Glucoamylase, also known as glucan 1,4-alpha-glucosidase, amyloglucosidase, gamma-amylase, lysosomal alpha-glucosidase, acid maltase, exo-1,4-alpha-glucosidase, glucose amylase, gamma-1,4-glucan glucohydrolase, acid maltase, and 1,4-alpha-D-glucan glucohydrolase, is an enzyme with a system name of 4-alpha-D-glucan glucohydrolase. The enzyme catalyzes the following chemical reaction: hydrolysis of terminal (1−>4)-linked alpha-D-glucose residues successively from non-reducing ends of the chains with release of beta-D-glucose. Most forms of the enzyme can rapidly hydrolyse 1,6-alpha-D-glucosidic bonds when the next bond in the sequence is 1,4. Genes that code for the expression of glucoamylase have been cloned into yeasts such as S. cervisiae. See, e.g., U.S. Pat. No. 5,422,267 to Yocum et al. Typically, glucoamylase is produced in industrial scale using microorganisms such as Aspergillus Niger. Often, glucoamylase is added to speed up fermentation of wort, honey, grape juice, or other fluids or solutions containing sugar.
As discussed in Pretorius et al. (1991), “The Glucoamylase Multigene Family in Saccharomyces cerevisiae var. diastaticus: An Overview,” CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 26(1):53-76, S. cerevisiae has been used widely both as a model system for unraveling the biochemical, genetic, and molecular details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and their substrate range in S. cerevisiae. The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by α- and βamylases, glucoamylases, and debranching enzymes. Starch utilization in the yeast S. cerevisiae var. diastaticus depends on the expression of the three unlinked genes, STA1 (chr. IV), STA2 (chr. II), and STA3 (chr. XIV), each encoding one of the extracellular glycosylated glucoamylases isozymes GAI, GAII, or GAIII, respectively. Additional research relating to research pertaining to S. cerevisiae can be found in Pugh et al. (1989), “Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae,” BIOCHIMICA ET BIOPHYSICA ACTA, 994: 200-209.
Despite the concerted and longstanding efforts of numerous researchers, a single organism capable of fermenting in an economically feasible manner biomass containing starch as the sole or main precursor to ethanol (or alcohol), e.g., without needing to add glucoamylase from another source, to replace hydrocarbon fuels such as gasoline has not been achieved. Although certain entities have strived to improve biomass biotechnological productivity, e.g., Mascoma Corporation (Lebanon, N.H.), none have achieved the level of success to meet long-felt industry needs as reflected by subject matter encompassed by the claims below.
Accordingly, there remains a need for such microorganisms and for methods of their preparation and use.